Materials and Methods Relating to the Induction of Apoptosis in Target Cells

ABSTRACT

Compositions, methods, uses and assemblages for the preferential induction of cell division cycle arrest and/or apoptosis, in a first population of cells compared to a second population of cells employ either: (i) an opioid or opioid-like agent and an NF-κB activating agent, which agents are other than the opioid-like agent trans-U50488 in combination with an NF-κB activating agent selected from etoposide and nocodazole; (ii) a ligand for a sigma receptor, or (iii) an opioid or opioid-like agent wherein the cells of the first population are other than tumour cells.

The present invention relates to materials and methods relating to theinduction of cell division cycle arrest and/or apoptosis in targetcells. In particular the target cells may be tumour cells or cellsinvolved in inflammatory disease processes.

BACKGROUND

It has been proposed that the cell's intrinsic apoptotic death programmeprovides a crucial restraint on tumourigenesis in at least 2 ways: (1)engagement of the apoptotic death programme in response tonon-rectifiable genetic damage prevents potentially oncogenic mutationsbeing passed to subsequent cell generations; (2) an inappropriatelyproliferating mass of cells engages its death programme when it beginsto outstrip its supply of exogenous diffusible and non-diffusiblesurvival signals. The restoration of a defective cell death programme istherefore a goal in tumour therapy.

International Patent Application WO 96/06863, describes opioid-likeagents for use in the induction of apoptosis. WO 96/06863 teaches thatthe behaviour of intracellular proenkephalin is influenced by survivalsignals which are dependent on cell density and in that way, a line ofcommunication is provided which informs the inside of the cell as to thesurvival conditions outside the cell. However, it was noted that whencultured rodent fibroblasts (3T3 cells) undergo spontaneoustransformation, intracellular proenkephalin immunofluorescence isdecoupled from cell density dependent signalling and indeedproenkephalin can be detected even at low cell densities. This behaviourof proenkephalin may allow the transformed cell to disregard the levelof external survival signals and so override the death programme in away beneficial to an early tumour cell mass. Experiments documented inWO 96/06863 showed that dysregulation of endogenous proenkephalin can bemimicked by cytoplasmic proenkephalin expressed from a heterologouspromoter. This prevents the cell entering into its death programme inresponse to agents which normally induce apoptosis. Thus cells whereproenkephalin is no longer subject to factors which regulate itsexpression or activity have a survival advantage which would allow anincipient tumour cell to withstand genetic damage and also to disregardits environment. Thus a pathway in which proenkephalin is involved is apotentially oncogenic pathway which may be widely involved intumourigenesis.

Cells in a non-transformed state require the continuous provision ofseveral types of survival signals. These are present in theextra-cellular environment in limiting amounts and are usually suppliedby neighbouring cells of different types. The survival signals suppressa cell's intrinsic death programme (Raff 1992 Nature Vol. 356 pp397-400). Thus, the abrogation of a single survival signal would not beanticipated to cause the death of the cell. However, in WO 96/06863 itwas taught that if incipient tumour cells acquire a survival advantagedue to autonomous upregulation of one pathway, then over a period oftime, the clonal mass of cells would become preferentially dependent onthat pathway for survival (due to a loss of selective pressure tomaintain alternative survival-promoting cell surface receptors, renderedredundant by a depletion of their own signalling molecules normallysupplied by cells or matrices of a different type). This was shown byadministration of monoclonal anti-proenkephalin antibodies to humancancer cells (in an attempt to abrogate proenkephalin-mediatedsurvival). The result was that apoptosis was induced in tumour cellswhereas non-transformed cell lines were less affected. This resultsupported the prediction that tumour cells would be more susceptible toabrogation of proenkephalin-mediated survival than non-tumour cells.

The identity of extracellular or intracellular proenkephalin receptorsis not yet known. However, proenkephalin can be proteolytically cleavedto enkephalin pentapapetides (Met- and Leu-enkephalin) which are knownto bind to delta opioid receptors. WO 96/06863 reports on the testing ofa synthetic opioid, naltrindole for induction of apoptosis in tumourcells and non-tumour cells. Naltrindole is an antagonist of the deltaopioid receptor, the same receptor to which enkephalins bind.Naltrindole was shown to potently induce apoptosis in many human tumourcells with non-tumour cells being less affected. This providedexemplification of preferential dependence of tumour cells onopioid-like pathways for survival.

The applicant also found that an agonistic ligand for the kappa opioidreceptor, trans-U50488, is a potent inducer of apoptosis in a wide rangeof tumour cells and once again, non-tumour cells are less affected.Synergy was also noted between the agents, naltrindole and trans-U50488.Trans-U50488 is a kappa ligand whereas cis-U50488 is a sigma ligand.

Pharmacologists originally subdivided opioid receptors into 4 mainclasses-mu, delta, kappa, and sigma (for review see Zukin, R. S andZukin S. R. Trends in Neurosciences 1984 pp 160-164). However, sigmareceptors were later regarded as distinct from opioid receptors and tobe viewed in a different class. Indeed, this was supported by thecloning of the mu, delta and kappa receptors which showed substantialhomology to each other. In contrast, the so-called type 1 sigma receptorshows no significant primary sequence homology to the opioid receptors(see for example Kekuda et al. 1996 Biochem Biophys res Commun Vol. 229,pp 553-558), although additional sigma receptor types remain to becloned. Evidence has recently emerged that, despite the lack of primarysequence homology, there is some cross-reactivity between sigma and themore “classical” opioid receptors. Kobayashi et al., 1996 Br J PharmacolVol 119, pp 73-80 report that sigma receptor ligands (such ascyclazocine, SKF-10047, and haloperidol) can interact with cloned mu-,delta-, and kappa-opioid receptors expressed in Xenopus oocytes. Howeverno evidence exists that sigma ligands interact directly with naturallyoccurring mu, delta or kappa opioid receptors. Conversely, fourdifferent kappa opioid receptor agonists (including trans-U50488, citedin WO96/06863) compete with a sigma-1 receptor ligand to the same extentas sigma-2 ligands (such as haloperidol and rimcazole) for sites inbrain, liver and spleen (Brent 1996 Brain Res Vol 725, pp 155-165). Amore detailed account of predicted interactions between opiate-relatedcompounds and sigma receptors is given by Walker et al (1990 PharmacolRev Vol. 44 pp 355-402). This indicates that among opioid-relatedcompounds, the determinants for sigma receptor activity differstrikingly from the determinants for opiate receptors; also, withopiate-related compounds, the (major subtype of) sigma receptor displaysreverse stereo selectivity to the classical opiate receptors.

Sigma receptors are known to be expressed on several different types oftumour cells. For example John et al. 1995 Life Sci Vol. 56, pp2385-2392, describes sigma receptors on human lung cancer cells; Brentand Pang 1995 Vol. 278, pp 151-160, describes sigma receptors on humanbreast and colon cancer cells and melanoma cells; Thomas et al. 1990Life Sci Vol. 46, pp 1279-1286, describes sigma receptors and opioidreceptors in human brain tumours. Vilner et al describe sigma receptorsin a wide variety of tumour cell lines (Vilner et al, 1995 Cancer Res 55408-413). Mach et al (Cancer Res 1997 57 156-161) suggest that sigmareceptors can be used as markers to assess the proliferative status of atumour.

Brent and Pang (1995, as above) described the inhibition of tumour cellproliferation by sigma ligands, as has been described for opioidreceptor ligands (see for example Maneckjee and Minna PNAS 1992 Vol 89pp 1169-1173). However, typically, proliferation results in a greaternumber of cells competing for available survival factors. Thusproliferation is associated with increased apoptosis and conversely aninhibition of cell proliferation is associated with reduced apoptosis.Indeed, Gerard Evan and colleagues proposed obligatory coupling of cellproliferation and cell suicide pathways (see for example Harrington etal Current Opinion in Genetics and Development 1994 Vol 4 pp 120-129.

Brent and colleagues have also described the induction of apoptosis by asigma receptor ligand, reduced haloperidol in colon and mammary celllines (Brent et al 1996 Biochem Biophys Res Commun Vol 219 pp 219-225).The natural opium alkaloid noscapine induces apoptosis in tumour cells(Ye et al. 1998 PNAS Vol. 95, pp 1601-16060.) Ye and colleagues alsoreport noscapine-induced regression of human tumour xenografts in micewith little evidence of toxicity and they propose that noscapinedepolymerises microtubules.

The transcription factor NF-κB has recently been linked to the controlof apoptosis. It is generally regarded as having an anti-apoptoticfunction. In most cell types, NF-κB is present in the cytoplasm in aninactive form bound to the inhibitor molecule IκB. NF-κB is activated bythe removal of the IκB molecule. (See Baeuerle and Baltimore 1996 CellVol. 87, pp 13-20 and Baichwal and Baeuerle 1997 Current Biology Vol. 7pp R94-R96).

Evidence of an anti-apoptotic role for NF-κB includes for example thepresence of massive liver cell apoptosis in RelA(p 65) (a subunit ofNF-κB) knock-out mice (Beg et al. 1995 Nature Vol. 376 pp 167-170).Furthermore, inactivation of NF-κB in B lymphocyte cell lines causesthem to apoptose (Wu et al. 1996 EMBO J. Vol. 15 pp 4682-4690). Theanti-apoptotic role of NF-κB has been proposed to reduce the efficacy ofanti-cancer therapies. Introduction of a “super-repressor” form of IκBαinto tumour cells prevents activation of NF-κB. This results in enhancedtumour cell killing in response to a number of anti-cancer agents (Wanget al. 1996 Science Vol. 274 pp 784-786; Antwerp et al. 1996 ScienceVol. 274 pp 787-789). Thus a number of cancer therapies including tumournecrosis factor (TNF) alpha, ionising radiation, daunorubicin, etoposideand vincristine are associated with undesirable activation of NF-κB. Begand Baltimore have gone so far as to deduce that NF-κB is an essentialelement in the prevention of TNF-α-induced death (1996 Science Vol. 274pp 782-784). As a result, there is much current interest in thepossibility that prevention of NF-κB activation will enhance tumour cellresponsiveness to a wide range of anti-cancer agents (see overview byBarinaga Science 1996 Vol. 274 p 724).

In some situations and cell types there is suggestion of a pro-apoptoticfunction of NF-κB. For example, serum starvation of 293 cells causescell death which is blocked by a dominant negative form of RelA (Grimmet al. 1996 J. Cell Biol. Vol. 134, pp 13-23) and also radiation-inducedapoptosis in fibroblasts from ataxia telangiectasia patients is reducedby “super-repressor” IκBα which prevents NF-κB activation (Jung et al1995 Science Vol. 268 pp 1619-1621). Thus, the relationship of NF-κB tothe control of apoptosis is unclear, with little immediate prospect ofunderstanding how a cell decides to engage anti- or pro-apoptoticoutcomes in response to NF-κB.

NF-κB has been linked to the genesis and progression of tumours. Forexample, disruption of IκBα leads to malignant transformation of 3T3cells (Beauparlane 1994 Oncogene Vol 9 pp 3189-3197) and the absence offunctional IκBα protein leads to constitutively active NF-κB in Hodgkinslymphoma lines (Wood et al. 1998 Oncogene Vol. 16, pp 2131-2139). Areciprocal relationship between NF-κB and the tumour suppressor p53 hasalso been shown (N. Perkins, unpublished). Constitutive activation ofNF-κB has also been linked with progression of breast cancer tohormone-independent growth (Nakshatri et al. Mol. Cell. Biol. 1997 Vol.17 pp 3629-3639). Thus the inappropriate activation of NF-κB in tumourspoints towards a therapeutic approach which neutralises or inhibits theactivity of NF-κB.

However, global abrogation of NF-κB activity would reduce its pro aswell as its anti apoptotic effects and would therefore be less effectivethan, for example, selective abrogation of the anti apoptotic effects.Further, none of the studies which describe an enhanced apoptotic effectof anti-cancer agents when NF-κB is inhibited, show a preferentialeffect on tumour compared to non-tumour cells (Barinaga Science 1996Vol. 274 p 724).

There is some evidence for a link between the NF-κB and opioid pathways.For example, NF-κB activates transcription of the proenkephalin gene inT lymphocytes (Rattner et al. 1991 Mol. Cell. Biol. Vol 11 pp1017-1022). The regulatory region of the delta opioid receptor gene alsocontains an element to which NF-κB would be expected to bind (Augustinet al 1995 Biochem Biophy Res Commun Vol. 207 pp 111-119).

Also, stimulation of the mu opioid receptor results in activation ofNF-κB in cultured neurons (Hou at al 1996 Neurosci Letts Vol 212 pp159-162). These results do not however suggest that opioids willmodulate the outcome of NF-κB activation.

Features shared by cells undergoing apoptosis and mitosis, such aschromosome condensation and nuclear lamina disassembly, led at one timeto the proposal that apoptosis is a form of aberrant mitosis (Earnshaw1995 Curr Op Cell Biol Vol 7 pp 337-343). This view was supported by anobservation of the necessity for active p₃₄ ^(cdc2) kinase, an enzymeuniversally required for entry into mitosis, in apoptotic death inducedby treatment with perforin and fragmentin-2 (Shi et al 1994 Science Vol263 pp 1143-1145). In contrast, other studies showed apoptosisunaccompanied by activation of p34^(cdc2) (Norbury et al. 1994 BiochemBiophys Res Commun Vol. 202-pp 1400-1406; Oberhammer et al 1994 J CellBiol Vol 126 pp 827-837). These conflicting data led to the conclusionthat the resemblance between apoptosis and mitosis is coincidental (seeEarnshaw review 1995).

On the other hand, a connection between decision-making events inproliferation and cell death control remains widely accepted (see reviewby Gerard Evan and colleagues: Harrington et al 1994 Curr Op Geneticsand Dev. Vol 4, pp 120-129). It has been proposed that an obligatecoupling between the cell death programme and cellular proliferationprovide a crucial brake on tumorigenesis. For example, c-Myc, a moleculeessential for cell proliferation, promotes cell death instead of cellproliferation when exogenous survival factors become limiting (Evan etal 1992 Cell vol 63 pp 119-125). A proliferating mass of cells whichoutstrips its supply of exogenous survival factors will die by apoptosisunless it has acquired the ability to survive by self-generated factors.This connection predicts therefore that a reduction in cellularproliferation will be accompanied by a reduction in the cell'spropensity to undergo apoptosis.

Apoptosis in response to opioid-like agents is preceded by an inductionof mitotic arrest (Ye et al PNAS 1998 Vol 95 pp 1601-1606). This is nota common pathway to apoptosis induction.

Evidence for this as an opioid-dependent mechanism of apoptosisinduction was provided in WO 96/06863 which disclosed that proenkephalinantibodies induced entry into mitosis but with failure of mitoticprogression. However, a connection between mitotic arrest and apoptosisinduction could not have been predicted at the time.

Activated NF-κB is proposed to play a role in both chronic and acuteinflammation (Baldwin 1996 Annu Rev Immunol Vol 14 pp 649-681). Forexample NF-κB is activated in arthritic synovium and anti-arthritictherapies block NF-κB activation. Acute inflammation such as septicshock is also associated with NF-κB activation. A number of chronicdiseases are not obviously inflammatory in nature, but inflammatorymechanisms may play a part. For example, autoimmune diseases such assystemic lupus erythematosus, atherosclerosis and Alzheimer's diseaseare all reported to be associated with NF-κB activation.

Haslett (1997 British Medical Bulletin Vol 53 pp 669-683) discoveredsome years ago that inflammatory cells undergo apoptosis constitutivelyand proposed that elimination of inflammatory cells by apoptosis is acrucial mechanism to limit the inflammatory response and that a failureof this mechanism leads to persistent inflammation. Molecules orpathways which inappropriately extend the lifespan of inflammatorycells, through delay in engagement of their constitutive deathprogramme, have yet to be identified.

The reduction of NF-κB activity is a goal of workers attempting todiscover therapies for inflammatory and associated disorders.

The present inventors have now discovered that ligands which bind opioidreceptors or relatives of opioid receptors induce apoptosispreferentially in tumour cells as compared to non-tumour cells, aproperty which was described in WO96/06863 for two named ligands,trans-U50488 and naltrindole. However, the present inventors now showthat these ligands induce apoptosis through an NF-κB-dependent mechanismand that this is at least partly consequent on arrest in mitosis(possibly through microtubule disruption). This NF-κB dependentmechanism, which involves switching activated NF-κB into a pro-apoptoticmode, has not been previously disclosed.

The results herein also show that the apoptotic effect which relies onactivated NF-κB is greater on tumour cells (particularly those whenNF-κB is constitutively active) than on non-tumour cells.

Prevailing scientific opinion indicates that the apoptotic effect ofanti-cancer agents is enhanced by the inhibition of NF-κB. In contrastthe present application demonstrates that opioid-mediated conversion ofactivated NF-κB from an anti- to a pro-apoptotic role can be used toenhance the efficiency of certain anti-cancer agents (e.g. TNF).

This approach is contrary to the prevailing scientific opinion in thefield.

In WO96/06863, a powerful cooperative effect (specifically, anacceleration of the onset of the death programme) between opioids andnocodazole was described. However, disclosure of the present applicationallows for the first time the skilled person to predict specificallythat agents which depolymerise microtubules (such as colchicine,nocodazole, podophyllotoxin and vinblastine, particularly since Ye et aldescribe noscapine binding sites on tubulin to be distinct) willcooperate with opioid-like compounds in apoptosis induction throughactivation of NF-κB.

The provision of a pro-apoptotic switch, to activated NF-κB, by a numberof opioid-like compounds, including noscapine, explains the apoptoticfate of cells in which microtubules have been depolymerised bynoscapine. However, the cooperation of opioids with NF-κB B to inducedeath is not reliant on microtubule depolymerisation as the stimulus toNF-κB activation.

The phenomenon of apoptosis preceded by mitotic arrest is shared bynoscapine and other opioid-like agents. Given that there is no universalconnection between apoptosis and mitotic arrest, this indicates thatopioid-like pathways, including those in which noscapine participates,are inducing apoptosis through a similar mechanism which is otherwiseuncommon.

Induction of apoptosis by opioid-like agents is not necessarilyconsequent upon mitotic arrest. Data herein for example with tumournecrosis factor (TNF), show that agents which induce activation of NF-κBin a microtubule-independent manner also cooperate with opioids toinduce apoptosis and that the cooperativity is blocked by IκB. Thus, thepro-apoptotic switch provided to activated NF-κB by opioid-like agentsis not exclusively dependent on microtubule depolymerisation effects.

The disclosure of Ye et al contains no suggestion of how noscapine maybe gaining access to the interior of the cell. Noscapine binds with highaffinity (in the nanomolar range) to brain-specific non-opioid sites(Mourey et al. 1992 Mol Pharmacol Vol. 42, pp 619-626) which leaves openthe question of what, if any, receptors noscapine is binding to on thesurface of tumour cells.

Kamei and colleagues (Kamei et al Eur J Pharmacol 1993 Vol. 242, pp209-211, and Kamei Pulm Pharmacol 1996 Vol. 9, 349-356) describe thatthe anti-tussive effects of noscapine are significantly reduced bypre-treatment with rimcazole, a specific antagonist of sigma sites. Theconclusion is that sigma sites may be involved in the anti-tussivemechanism of non-narcotic anti-tussive drugs (such as noscapine anddextromethorphan). This data suggests some indirect cross talk betweenthe two receptor systems, rather than direct cross reaction of noscapinewith sigma receptors.

However, the high dose of noscapine (120 mg/kg by intraperitonealadministration to mice) described by Ye et al to be required forinduction of apoptosis and tumour regression is substantially higherthan the dose required (10 mg/kg intraperitoneally) to produce ananti-tussive effect. From this teaching the skilled person could notdeduce that the anti-tumour effect of noscapine is mediated throughsigma receptors on the cell surface. Indeed, Ye and colleagues show anintracellular microtubule depolymerising effect of noscapine, due, theypropose, to a fortuitous structural similarity between noscapine,colchicine and podophyllotoxin which are known microtubuledepolymerising agents and have no known affinity for cell surface orintracellular opioid or sigma receptors.

This application shows that noscapine, surprisingly, shares the samemechanism of apoptosis induction in tumour cells as opioid receptorligands and sigma receptor ligands. The inventors now demonstrate thattumour cells in which NF-κB is constitutively activated are particularlysusceptible to noscapine. They also demonstrate that noscapinecooperates with exogenous activators of NF-κB in tumours where NF-κB isnot known to be constitutively active.

It is known that opioid receptors can be internalised after ligandbinding by an endocytic mechanism. In this way, ligands can be deliveredto the interior of the cell (Keith et al. 1998 Mol. Pharmacol. Vol 53,pp 377-384). The present inventors propose that noscapine is gainingaccess to the interior of the cell, and thence to microtubules, afterbinding to opioid-like receptors on the cell surface. Microtubuledepolymerisation is a known stimulus to NF-κB activation (Rosette andKarin 1995 J. Cell Biol. Vol 128 6 pp 1111-1119) but this would not initself be a sufficient explanation for apoptosis induction by noscapinesince NF-κB activation generally provides an anti-apoptotic effect (seebelow). The present application shows that noscapine, opioid and sigmaligands use the same, otherwise uncommon, mechanism to induce apoptosis.Namely the provision of a pro-apoptotic switch to activated NF-κB atleast partly through microtubule depolymerisation.

The present application explains why noscapine has an unexpectedpro-apoptotic effect, since it provides a pro-apoptotic switch toactivated NF-κB. In the examples it is shown that blockade of NF-κBactivation by IκB blocks noscapine and other opioid-induced death. Theinventors show that proliferating non-tumour cells retain correct cellcycle checkpoint control in the present of opioids (seeexemplification). This provides a further mechanism for enhancedtoxicity in tumour compared to non-tumour cells.

The present invention also addresses the role of sigma ligands ininducing apoptosis in tumour cells and provides evidence that suchligands employ the same novel NF-κB dependent mechanism of apoptosisinduction as conventional opioid receptor ligands. Walker et al (1990Pharmacol. Rev. Vol. 42 pp 335-402) comment that the sigma receptordisplays reverse stereo selectivity to the classical opioid receptors.Of particular relevance, cis-isomers of U50488 bind to sigma receptorswhereas trans-isomers of U50488 bind to kappa receptors. In theexperiments of WO96/06863, the trans-isomer of U50488 (Sigma ChemicalCompany D-8040;trans-3,4-dichloro-N-methyl-N-(2[1-pyrrolidinyl]cyclohexyl)-benzeneacetamide)which binds kappa receptors in preference to sigma receptors, is used.Different isomers of benzomorphans also prefer to bind either kappa orsigma receptors. Thus, in two unrelated classes of compounds, differentisomers show preferences for kappa or sigma receptors. Walker commentsthat this suggests a possible relationship between the topography of thekappa opiate and sigma receptor binding sites. A cooperative functionalinteraction between kappa and sigma binding sites is taught herein. Itis shown that the sigma ligand rimcazole cooperates with a trans-isomerof U50488 (a kappa ligand) in the induction of apoptosis in cancer cells(FIG. 10 b).

Brent et al (1996 Brain Res Vol 725, pp 155-165) describe that kappaopioid receptor agonists, including trans-U50488, moderately inhibitsigma-1 receptor binding in guinea pig brain, liver and spleen. However,Brent present no evidence that kappa agonists interact directly withsigma receptors. An explanation for his data would be that kappareceptors are coupled, perhaps by heteroligomerisation, to sigmareceptors. Another explanation would be that a distinct kappa-likebinding pocket is allosterically coupled to a sigma binding pocket,within the same receptor macromolecule. In that way, kappa ligands couldindirectly affect the binding of specific sigma ligands. Furthermore,there is no evidence presented by Brent that kappa ligands affect thebinding of sigma ligands to human tumour cells. In the presentapplication the role of sigma ligands in inducing apoptosispreferentially in tumour compared to non-tumour cells is specificallyaddressed and it is shown that sigma ligand-induced apoptosis ismediated at least in part by the same intracellular NF-κB-dependentevents as more conventional opioid receptor ligands. The elucidation ofthis common mechanism has allowed the present inventors to predict, andexemplify, that apoptosis is preferentially induced in tumour comparedto non-tumour cells. Tumour cells in which NF-κB is constitutivelyactivated are particularly susceptible; otherwise, the sigma ligands canbe co-administered with exogenous activators of NF-κB, as is proposedfor opioid receptor ligands.

Brent and colleagues have also described the induction of apoptosis by asigma receptor ligand, reduced haloperidol, in colon and mammarycarcinoma cell lines (Brent et al 1996 Biochem Biophys Res Commun Vol.219 pp 219-226). The demonstration of apoptosis induction per se doesnot in itself predict application of an agents to anti-cancer therapysince there are many hundreds of agents which induce apoptosis in cellsbut do so to the same extent in diseased compared to non-diseased cells;hence, any application to an in vivo situation would be predicted to befatal. Thus, a crucial feature of an apoptosis-inducing agent of likelytherapeutic ability is a clear preference to induce apoptosis indiseased compared to non-diseased cells. This is shown in the presentapplication. A more recent paper on the potential applicability of sigmaligands to cancer management is in the area of diagnostics rather thantherapy. Specifically, Mach and colleagues (Cancer Res. 1997 Vol. 57 pp156-161) acknowledge that the function of sigma receptors on tumourcells is unknown, but suggest that sigma ligands may be used as markersto assess the proliferative status in tumours. No suggestion is madethat they may be therapeutically important in their own right.

Opioids are also beneficial in providing a pro-apoptotic switch toactivated NF-κB in chronically inflamed cells, thereby contributing to aresolution of the inflammatory process. An inappropriate NF-κB mediateddrive to survive in chronic inflammation, which is apparent through theextended average life span of inflammatory cells, leads to a loss ofselective pressure to maintain alternative survival pathways oversuccessive inflammatory cell generations. Inflamed cells under theseconditions become locked into a dependence on one pathway for survival,in an analogous way to tumour cells. Global inhibition of NF-κB activityis one potential therapeutic strategy because it would be more effectivein chronically diseased cells which have been reliant on NF-κB forsurvival. However, the duplicitous nature of NF-κB means that the effectof this approach might be offset by a reduction in an associated NF-κBmediated pro-apoptotic drive (i.e. chronically inflamed cells also existon a knife-edge between life and death in the same way proposed fortumour cells). An alternative and entirely novel strategy wouldtherefore be to provide a pro-apoptotic switch to activated NF-κB usingopioid-like pathways. This strategy would not necessarily rely on thepresence of opioid receptors on inflammatory cells since therapies canemploy agents linked to internalisation peptides such as “Penetratin” aswas described in WO96/05863.

According to a first aspect of the present invention there is provided acomposition for the preferential induction of cell division cycle arrestand/or apoptosis, in a first population of cells compared to a secondpopulation of cells which composition comprises an opioid or anopioid-like agent and an NF-κB activating agent, which agents are otherthan the opioid-like agent trans-U50488 in combination with an NF-κBactivating agent selected from etoposide and nocodazole.

The cells of the first population, compared to cells of the secondpopulation, may be referred to herein as “abnormal” and/or “undesirable”cells. The cells of the second population are preferably normal and/ordesirable cells within the context of the intended use of the invention,for example cells which do not display characteristics typicallyassociated with a disease or condition which is intended to be treatedusing the invention. In particular the cells of the first population arepreferably tumour cells or undesirable cells of an inflammatory processand the cells of the second population are preferably non-tumour and/ornon-inflammatory cells.

The phrase “cell division cycle arrest” is intended to mean that thecells in the population fail to increase in number over time.

The abnormal and/or undesirable cells may in some instances have apreferential dependance for survival on the pathways in which productsof opioid peptide precursor and/or sigma receptor genes participate.

An opioid-like agent is a ligand which is capable of binding to one ormore receptors of the mu opioid receptor, the delta opioid receptor, thekappa opioid receptor or the sigma receptor class or to another receptorcapable of binding known ligands for the mu, delta, kappa and sigmareceptors. Thus an opioid-like agent may be a ligand as stated abovewhich can bind one or more of said receptors with moderate to highaffinity defined according to standard pharmacological principles (seefor example review by Walker et al., 1990 Pharmacologica Reviews Vol. 42pp 355-400).

Examples of opioid-like agents according to the present inventioninclude proenkephalin; derivatives of proenkephalin (such as met- andleu-enkephalin); noscapine; kappa receptor agonists (such astrans-U50488, bremazocine, spirodoline, ICI 197067, (−)-pentazocine,(−)-ethylketocyclazocine, (−)-cyclazocine and (−)-N-allylnormetazocine((−)-SKF10,047)); delta receptor antagonists (such as naltrindole); andsigma receptor ligands (such as haloperidol, reduced haloperidol,rimcazole, 1,3-di (2-tolyl) guanidine, (+)-N-allyl normetazocine,(+)-pentazocine, (+)-ethylketocyclazocine, (+)-benzomorphans such as(+)-pentazocine and (+)-ethylketocyclazocine, (+)-morphinans such asdextrallorphan, cis-isomers of U50488 and analogues, arylcyclohexaminessuch as PCP, N-N′-diryl-substituted guanidines such as DTG,phenylpiperidines such as (+)-3-PPP and OHBQs, steroids such asprogesterone and desoxycorticosterone, butryophenones, BD614,(+/−)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolodinyl)cyclohexylamine,antipsychotic and potential antipsychotic drugs, additional tohaloperidol and rimcazole, which bind with a moderate to high degree ofpotency to sigma sites including: perphenazine, fluphenazine,(−)-butaclamol, acetophenazine, trifluoperazine, molindone, pimozide,thioridazine, chlorpromazine and triflupromazine, BMY 14802, BMY 13980,remoxipride, tiospirone, cinuperone (HR 375), WY47384; antidepressantsincluding amitriptyline and imipramine; see e.g. Walker et al 1990Pharmacological Reviews Vol. 42 p 355-400).

The above mentioned compounds are exemplary opioid-like agents. Othersmay be readily ascertained by those skilled in the art, based on theabove described binding characteristics.

Generally speaking kappa agonists are those agents which preferentiallystimulate activity at kappa opioid receptors when tested against otheropioid receptor types. Delta antagonists are those agents whichpreferentially antagonise the activity of delta opioid receptors whentested against other opioid receptor types. Sigma ligands are thoseagents which preferentially bind to sigma receptors when tested againstother opioid receptor types. A ligand for a sigma receptor for use inaccordance with the invention can be identified by the following method(Vilner et al Cancer Res 1995 55:2 408-413).

Firstly, a suitable preparation such as a crude membrane portion ismade, by conventional protocols, from a cell type, such as a humantumour cell line, which is known to express sigma receptors. Examples ofsuch cell lines would include; A375 melanoma (Accession No: ECACC88113005), SK-N-SH neuroblastoma (Accession No: ECACC 86012802) andLNCaP.FGC prostate (Accession No: ECACC 89110211). These cell lines areobtainable from the European Collection of Animal Cell Cultures (PortonDown, England) with reference to the accession numbers shown.

The binding of a putative sigma ligand to sites on these preparations isthen measured in comparison to the prototypic sigma ligands such as(+)-pentazocine and 1,3-di-o-tolylguanidine (DTG) (and as described byWalker et al., 1990 Pharmacologica Reviews Vol. 42 pp 355-400). Radio orchemically labelled prototype sigma ligands are allowed to bind to sigmareceptors in the cell preparation. The amount of labelled prototypesigma ligand displaced by the putative ligand is measured and used tocalculate the affinity of the putative ligand for the sigma receptor.

An NF-κB activating agent is any agent capable of activating NF-κB.Examples of NF-κB activators suitable for use according to the presentinvention can include; cytokines, mitogens, prostaglandins,leukotrienes, bacteria, bacterial proteins, viruses, viral proteins,chemical agents, oxidising agents, microtubule depolymerising agents(such as colchicine, nocodazole, podophyllotoxin and vinblastine),genotoxins (such as etoposide) and the RelA(p65) subunit of NF-κB oragents which cause its expression, overexpression or activation (e.g. anexpression vector for RelA(p65) or a transcription factor which causesits upregulation or an agent which induces its translocation to the cellnucleus). Further exemplary agents are named in the following table(From Siebenlist et al 1994 Annu. Rev. Cell Biol. 10 405-455).

Cytokines Tumour necrosis factor-α (TNF-α) Lymphotoxin (LT) (TNF-β)Interleukin-1 α and β (IL-I α and β) Interleukin-2 (IL-2) LeukemiaInhibitory factor (LIF) (Interferon-γ) (Macrophage colony-stimulatingfactor (M- CSF) (Granulocyte/macrophage colony-stimulating factor)(GM-CSF) Mitogens Antigen Allogenic Stimulation Lectins (PHA, Con A)anti-αβ T cell receptor anti-CD3 anti CD2 anti-CD28 Phorbol esters(Diacylglycerol (DAG) Calcium ionophores (ionomycin, A2837) anti-surfacelgM (P39) (CD-40 ligand) Serum (Platelet-derived growth factor) (PDGF)Other Leukotriene B4 biological (Prostaglandin E2 (PGE2) mediators(Insulin) Bacteria and Shigella flexneri bacterial Mycobacteriumtuberculosis products Cell wall products: Lipopolysaccharide (LPS)Muramyl peptides (G(Anh)MTetra) Toxins: Staphylococcus enterotoxin A andB (SEA and SEB) Toxic shock syndrome toxin-1 (TSST-1) (Cholera toxin)Viruses and Human T cell leukemia virus-1 (HTLV-1) viral Tax productsHepatitis B virus (HBV) Hbx MHBs Epstein-Barr virus (EBV) EBNA-2 LMPCytomegalovirus (CMV) (Human immunodeficiency virus-1) (HIV-1) Humanherpes virus-6 (HHV-6) Newcastle disease virus Sendai virus Adenovirus 5ds RNA Eukaryotic Theileria parva parasite Physical UV light stressIonizing radiations (X and γ) (Photofrin plus red light) (Hypoxia)Partial hepatectomy Oxidative Hydrogen peroxide stress Butyl peroxideOxidised lipids (Antimycin A) Chemical Calyculin A agents Okadaic acid(Pervanadate) (Ceramide) (Dibutyrl c-AMP) (Forskolin) Protein synthesisinhibitors Cycloheximide Anisomycin Emetine

The composition may be in the form of a pharmaceutical. The compositionmay be for the preferential induction of cell division cycle arrestand/or apoptosis, in abnormal and/or undesirable cells of a particulartissue type, in which case the NF-κB activating agent may have therequisite tissue specificity. For example the NF-κB activator CD40ligand is specific for lymphoid tissue.

Pharmaceutical compositions according to the present invention, mayinclude, in addition to active ingredient, a pharmaceutically acceptableexcipient, carrier, buffer, stabilised or other materials well known tothose skilled in the art. Such materials should be non-toxic and shouldnot interfere with the efficacy of the active ingredient. The precisenature of the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. cutaneous,subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

The present invention also provides a method for the preferentialinduction of cell division cycle arrest and/or apoptosis, in a firstpopulation of cells compared to a second population of cells, whichcomprises exposing cells to an opioid or an opioid-like agent and anNF-κB activating agent, which agents are other than the opioid-likeagent trans-U50488 and an NF-κB activating agent selected from etoposideand nocodazole. The populations of cells may be as stated above.“Opioid-like agent”, and “NF-κB activating agent” are as defined andexplained above.

The method may be employed ex vivo or in the treatment of a patient, inwhich case pharmaceutical composition according to the invention may beadministered to the patient. Alternatively the patient may be treatedwith ionising radiation or U-V light (see table) to activate NF-κB intarget cells combined with administration of a pharmaceuticalcomposition comprising an opioid or opioid-like agent.

The method may be for the preferential induction of cell division cyclearrest and/or apoptosis, of cells of a particular cell type, in whichcase use may be made of an NF-κB activating agent which has therequisite tissue specificity. For example for preferential induction ofcell division cycle arrest and/or apoptosis, in abnormal and/orundesirable cells in lymphoid tissue, CD40 ligand may be employed as theactivator of NF-κB.

Administration of a pharmaceutical composition according to theinvention or radiation/U-V light and a pharmaceutical compositioncomprising an opioid or opioid-like agent is in a “prophylacticallyeffective amount” or a “therapeutically effective amount” (as the casemay be, although prophylaxis may be considered therapy), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors.

The treatment in accordance with the present invention may be givenalone or in combination with other treatments, either simultaneously orsequentially dependent upon the condition to be treated.

The present invention also provides a method for the preparation of acomposition for the preferential induction of cell division cycle arrestand/or apoptosis, in a first population of cells compared to a secondpopulation of cells which method comprises selecting an agent for itsability to activate NF-κB and combining this NF-κB-activating agent withan opioid or opioid-like receptor ligand. The present invention alsoprovides a method for the preferential induction of cell division cyclearrest and/or apoptosis, in a first population of cells compared to asecond population of cells which comprises selecting an agent for itsability to activate NF-κB and exposing cells to this NF-κB activatingagent and to an opioid or opioid-like receptor ligand. The cells,“opioid-like agent” and “NF-κB activating agent” are all as stated andexplained above. A tissue-specific activating of NF-κB may be selected.

The present invention also provides use of an NF-κB activating agent andan opioid or opioid-like receptor ligand in the preparation of acomposition for the preferential induction of cell division cycle arrestand/or apoptosis, in a first population of cells compared to a secondpopulation of cells. The use may be in relation to the preparation of apharmaceutical composition. The use may be in relation to thepreparation of a pharmaceutical composition for the treatment oftumours. The use may be in relation to the preparation of apharmaceutical composition for the treatment of undesirableinflammation.

Similarly the present invention provides use of an NF-κB activatingagent and an opioid or opioid-like receptor ligand in the design of atreatment regime for the preferential induction of cell division cyclearrest and/or apoptosis, in a first population of cells compared to asecond population of cells. The treatment regime may be in relation tothe treatment of tumours or undesirable inflammation. The use may be ofa tissue-specific NF-κB activating agent.

The present invention also provides an assemblage of a composition asdisclosed above with direction instructing administration of thecomposition in a manner which results in the preferential induction ofcell division cycle arrest and/or apoptosis, in a first population ofcells compared to a second population of cells. Thus the presentinvention provides an assemblage of a composition as disclosed abovewith directions instructing administration of the composition to apatient with or at risk of a tumour or to a patient with or at risk ofan undesirable inflammation.

In some instances the cells of the first population may in terms offunction be p53 null tumour cells. In other instances the cells of thefirst population may in terms of function be p53 wild-type tumour cells.Compositions, methods and uses as set forth above which employ an NF-κBactivating agent and an opioid or an opioid-like receptor ligand andcomposition, methods and uses as set forth above which employ a ligandfor a sigma receptor may also employ p53 or an agent which causesexpression, overexpression and/or activation of p53 (such as anexpression vector for or agent causing upregulation of p53). The presentinventors demonstrate herein powerful cooperativity between sigmaligands and p53 using three different systems. Furthermore, the sigmaligands are effective in the nanomolar range, providing strong evidencethat the apoptotic effect is mediated through authentic sigma receptors.

According to another aspect of the present invention there is provided acomposition for the preferential induction of cell division cycle arrestand/or apoptosis, in a first population of cells compared to a secondpopulation of cells, which composition comprises a ligand for a sigmareceptor. “Sigma receptor ligands” are as defined and explained above.The cells are as stated above and may be tumour cells or undesirablecells of an inflammatory process. Examples of sigma receptor ligands arestated above. The composition may be in the form of a pharmaceutical.The pharmaceutical may take a general form as described above.

The present invention also provides a method for the preferentialinduction of cell division cycle arrest and/or apoptosis, in a firstpopulation of cells compared to a second population of cells whichcomprises exposing cells to a ligand for a sigma receptor. The methodmay be employed to treat a patient or may be ex vivo. The method maycomprise the administration of a pharmaceutical composition as providedabove wherein the effective agent is a sigma receptor ligand. Thepresent invention also provides a method for the preparation of acomposition for the preferential induction of cell division cycle arrestand/or apoptosis, in a first population of cells compared to a secondpopulation of cells which method comprises incorporating a ligand of asigma receptor into the composition. The method may comprise thepreparation of a pharmaceutical composition.

The present invention also provides an assemblage of a composition asabove comprising a ligand for a sigma receptor with directionsinstructing administration of the composition in a manner which resultsin the preferential induction of cell division cycle arrest and/orapoptosis, in a first population of cells compared to a secondpopulation of cells. The instructions may direct administration of thecomposition to a patient with or at risk of a tumour as to a patientwith or at risk of an undesirable inflammation.

Compositions, methods and uses as set forth above which employ a ligandfor a sigma receptor may also employ as an effective agent an agonistfor a kappa receptor. Kappa agonists are as stated and explained above.Exemplary kappa agonists are also mentioned above.

Composition, methods and uses as set forth above may be employed inrelation to situations e.g. medical conditions/disease statescharacterised by dysfunctional regulation of NF-κB, the dysfunctionalregulation being characterised by one or more of the following: thepresence of constitutively activated NF-κB in cells where it would notnormally be found, activation of NF-κB to supranormal levels and/or withan abnormally long duration of activation following cellularstimulation, and the presence of activated NF-κB which has an abnormalsubunit composition.

The present invention also provides a composition, and correspondingmethods and uses as set forth for previous aspects of the invention, forthe preferential induction of cell division cycle arrest and/orapoptosis, in a first population of cells compared to a secondpopulation of cells which composition comprises an opioid or anopioid-like agent (i.e. an NF-κB activating agent need not be present)wherein the cells of the first population are other than tumour cells.Such cells may be characteristic of an inflammatory disease (ie adisease where inflammation plays a role), arthritis, atherosclerosis,Alzheimer's disease, multiple sclerosis, autoimmune disease.

The cells of the first population may in some such instances have apreferential dependance for survival on the pathways in which productsof opioid peptide precursor and/or sigma receptor genes participate.

Where more than one agent or ligand as defined herein is used inaccordance with the invention in any of its aspects, at least one andpreferably all is/are present or used in a sublethal amount, i.e. anamount insufficient for that ligand or agent alone to induce inhibitionof the cell division cycle and/or apoptosis in a substantial proportionof target cells such that those cells fail to recover viability. Suchuse can have the advantage of reduced toxicity of the agents or ligandsto non-target cells.

Appendix 1 at the end of the examples provides IUPAC chemical namesfor 1) named agents which induce apoptosis and 2) prototypic sigmaligands for assays to ascertain sigma agents.

FIGURES

FIG. 8 illustrates a representative FACS (fluorescence activated cellsorting) profile obtained from the experiment indicated; horizontal axisdenote DNA content of the cells and the vertical axis the numbers ofcells that have a particular DNA content. Apoptotic cells have asubnormal DNA content and are represented as a peak to the extreme leftof the profile (sub-G1); viable cells are represented in G1 and G2/Mpeaks which correspond to these phases of the cell cycle. Wherepercentages are provided, these indicate cells in sub-G1, G1 or G2/Mphases, as indicated, as a percent of the total cell population. In allcases, the existence of apoptotic cells was confirmed by additionalmeans such as microscopic examination.

Bar charts show quantitative data obtained from representative FACSanalyses for the experiments indicated. In all cases, numbers of cellsthat are apoptotic (sub-G1), as a percentage of the total cellpopulation, are depicted by diagonal-hatched bars; numbers of cells thatare viable and in G1 (G1/S) phase of the cell cycle, as a percentage ofthe total cell population, are depicted by stippled bars; viable cellsin G2/M phase of the cell cycle are depicted by horizontal-hatched bars.

FIG. 1

Quantitative representation of FACS analysis showing cooperation ofopioids (Naltrindole) with TNF in apoptosis induction and its diminutionwhen NF-κB is inhibited by IκB.

FIG. 2

Quantitative representation of FACS analysis showing the cooperation ofproenkephalin with TNF in NFκB-dependent apoptosis induction in 293cells.

FIG. 3

Quantitative representation of FACS analysis showing cooperation betweenNoscapine and TNF in apoptosis induction in p53 null lung carcinoma(H1299) cells.

FIG. 4

Quantitative representation of FACS analysis showing cooperation ofNoscapine and TNF in inducing apoptosis in human colon carcinoma (HT29)cells.

FIG. 5

Phase micrographs of 293 cells transiently transfected with a DNAexpression vector encoding nuclear proenkephalin showing apoptoticinduction in combination with the p65 (RelA) subunit of NF-κB and moremarkedly when proenkephalin is in triple combination with p65(RelA) andp53.

FIG. 6

Quantitative representation of FACS analysis of 293 cells showingapoptotic induction with trans-U50488 and Naltrindole alone, andsynergistically when in combination; and a marked reduction inopioid-induced apoptosis with a repressor of NF-κB (IκB).

FIG. 7 a

Quantitative representation of FACS analysis of L428 Hodgkins lymphomacells showing induction of apoptosis at 0.1 mM Naltrindole and 0.1 mMNoscapine and the induction of apoptosis and cell cycle (G2/M) arrest at0.01 mm Naltrindole and 0.01 mM Noscapine.

FIG. 7 b

Quantitative representation of FACS analysis of Control B lymphoid cellsshowing the absence of any effect of Naltrindole at 0.1 mM or 0.01 mM orNoscapine at 0.1 mM or 0.01 mM, compared to untreated cells.

FIG. 8

FACS profile of H1299 cells treated with the delta receptor antagonistNaltrindole and TNF, showing cell cycle (G2/M) arrest prior toapoptosis.

FIG. 9 a

Quantitative representation of FACS analysis of (p53 null) H1299 humanlung carcinoma cells treated with haloperidol and TNF showingsynergistic apoptotic effect.

FIG. 9 b

Quantitative representation of FACS analysis of 293 cells showingsynergistic cooperation between haloperidol and the NF-κB subunitp65(RelA) in inducing apoptosis.

FIG. 9 c

Quantitative representation of FACS analysis of 293 cells showing thatapoptosis induced by haloperidol, a sigma receptor ligand, is dependenton NF-κB (in these cells which have functionally inactivated p53).

FIG. 10 a

Quantitative representation of FACS analysis of p53 null lung carcinoma(H1299) cells showing cooperation between the sigma receptor ligandrimcazole and TNF in inducing apoptosis.

FIG. 10 b

Quantitative representation of FACS analysis of H1299 cells showingcooperation between the sigma receptor ligand and rimcazole and thekappa receptor agonist trans-U50488 in the induction of apoptosis.

FIG. 11 a

Quantitative representation of FACS analysis of L428 (Hodgkins lymphoma)cells showing apoptosis induction by haloperidol at 0.1 mM, 0.01 mM and0.001 mM.

FIG. 11 b

Quantitative representation of FACS analysis of L428 (Hodgkins lymphoma)cells showing apoptosis induction by rimcazole at 0.1 mM, 0.01 mM and0.001 mM.

FIG. 11 c

Quantitative representation of FACS analysis of control B lymphoid(Daudi) cell line which are unaffected by rimcazole at 0.1 mM, 0.01 mMand 0.001 mM.

FIG. 12

Quantitative representation of FACS analysis of hormone responsive(MCF7) and hormone unresponsive (MDA MB 231) human breast carcinomacells showing a greater propensity of rimcazole to induce apoptosis andcell division cycle arrest in the hormone unresponsive (advanced stage)breast cancer cells.

FIG. 13

FACS analysis of hormone insensitive (advanced) breast cancer (MDA MB468) cells showing apoptosis induction by rimcazole alone, andsynergistic apoptosis induction when two sigma ligands are combined:rimcazole and cis-U50488.

FIG. 14 a

Phase micrographs of MCF 7 breast cancer cells expressing a conditionalmutant form of p53 which is inactive at the restrictive temperature of37 degC. (upper panels) but is activated if the temperature of theculture is reduced to 32 degC. (lower panels); rimcazole atconcentrations of 0.01 mM and below induces apoptosis to a substantiallygreater extent when p53 is activated in these cells by temperaturereduction.

FIG. 14 b

Quantitative representation of FACS analysis of human osteosarcoma(Saos-2::p53^(teti)) cells treated with low dose rimcazole and thetetracycline analogue doxycycline (to induce stably transfected p53driven by a tetracycline inducible promoter); this shows synergisticapoptosis induction when the two agents are combined.

FIG. 14 c

Quantitative representation of FACS analysis of 293 cells transientlytransfected with p53 cDNA and treated with rimcazole; this illustratessynergistic apoptosis induction when the two treatments are combined.

FIG. 15

Shows the results of xenograft studies using the MDA MB 468 human breastcarcinoma cell line in mice in the presence or absence of the sigmareceptor ligands rimcazole, cis-U50488 and haloperidol. FIG. 15 a showsmean tumour volume versus time; FIG. 15 b shows mean excised tumourweights and FIG. 15 c shows mean excised tumour weights for selectedtumours. All show some reduction of tumour growth in the presence of thesigma ligands, especially rimcazole.

The experiments described in the examples were performed using methodswell-known to those skilled in the art and documented in standard textbooks in the field (e.g. Chapters 4 and 5, Methods in Cell Biology Vol46 editors L. M. Schwartz and B. A. Osborne, Academic Press Ltd, London1995 provides inter alia details on detection of apoptosis and celldivision cycle arrest by FACS).

The apoptotic effect of TNF is known to be offset by NF-κB activation(Beg and Baltimore 1996 Science Vol. 274 pp 783-784; Wang et al. 1996Science Vol. 274 pp 784-787; Van Antwerp et al 1996 Science Vol. 274 pp787-789). In this experiment we addressed whether opioid-like agentswould enhance the apoptotic effect of TNF.

1.1 Opioid Receptor Ligands Enhance Tumour Necrosis Factor (TNF)-InducedDeath by Provision of a ‘Pro Apoptotic Switch to Activated NF-κB

FIG. 1 shows a quantitative representation of FACS analysis of 293 cells(transformed kidney epithelial cells) using the nucleic acid dye,propidium iodide; this gives a measure of DNA content in the cell. Thistechnique allows a simultaneous assessment of the cell division cycleprofile of cells as well as the presence of apoptotic cells. Viablecells (as a percentage of the total cell population) in G1/S or G2/Mphase of the cell cycle are represented as stippled orhorizontal-hatched bars. Apoptotic cells have a reduced DNA content dueto endonuclease activation which occurs during execution of theapoptotic programme; apoptotic cells (as a percentage of the total cellpopulation) are depicted as a sub-G1 population (diagonal-hatched bars).Untreated or TNF-treated cells (FIGS. 1 and 2) have very low levels ofapoptosis (2-4%). Treatment of cells with Naltrindole induces 46% ofcells to become apoptotic at this time point (approximately 48 hoursafter treatment addition); when naltrindole and TNF are combined, thereis a marked increase in apoptosis to 86%, which is reduced to 15% in thepresence of a repressor of NF-κB activity (IκB).

Prevailing scientific opinion would indicate that the most obviousexplanation for cooperation between opioid receptor ligands and TNF isthat naltrindole is inhibiting NF-κB activation. We were thereforesurprised to find that when a dominant inhibitor of NF-κB activation, anon-degradable form of IκB, was transfected into cells, the apoptoticpeak was markedly reduced and there was reappearance of G1 viable cells,indicating that death was largely abrogated (FIGS. 1 a and b). The fewremaining dead cells were likely to reflect the non-transfectedpopulation since it is rare to achieve 100% transient transfectionefficiency. These data therefore indicate that in this cell system(which is functionally p53 null) the cooperation between naltrindole andTNF requires NF-κB activation. Data from other laboratories has shownthat TNF activates an NF-κB mediated drive to survive which is inhibitedby IκB. Inhibition of NF-κB blocks the cooperativity between opioids anda non-lethal dose of TNF. We therefore propose that opioid pathwaysprovide a pro-apoptotic switch to activated NF-κB which may be operatingin one of the following ways:

1. Opioids selectively inhibit the anti-apoptotic arm of NF-κB activitybut spare its pro-apoptotic effects; in this way balance is shiftedtowards death. In non-transformed cells, abrogation of a single survivalpathway would not be expected to cause the death of a cell. In tumourcells, an acquired preferential dependence on NF-κB-mediated survivalwould render the tumour cell preferentially susceptible. Opioids are notproviding a global inhibition of NF-κB activation since transfection ofIκB alone has no effect on viability.

2. Opioids selectively promote the pro-apoptotic mediators downstreamfrom NF-κB; in this way, the balance would be shifted towards death andagain, we would anticipate that tumour cells would be preferentiallyaffected due to the tumour cell being poised on a knife-edge betweenlife and death, for the reasons discussed previously.

3. A combination of the above.

4. Any of the mechanisms described in 1-3, which are reliant on NF-κBactivation, may be implemented by an indirect effect of opioids onheterologous transcription factors; a number of these cooperate withNF-κB and play an important role in specifying NF-κB-responsive genetargets (Perkins, 1997, Int. J. Biochem. Cell Biol. Vol. 29 pp1433-1448).

It is interesting that a non-lethal dose of TNF (i.e. one which has noapoptotic effect on its own) cooperates powerfully with opioid-likereceptor ligands and this would not have been anticipated. At this doseof TNF we propose that an NF-κB-mediated anti-apoptotic drive outweighsits death agonistic effects. The use of a very low dose of TNF makes thecombination with opioids a particularly attractive therapeuticpossibility as the known harmful side effects of TNF will be greatlyreduced. The cooperation with TNF is seen with all opioid-like compoundswhich induce apoptosis: these include naltrindole, trans-U50488,noscapine and sigma receptor ligands including haloperidol andrimcazole.

1.2 Secretory Proenkephalin Cooperates with TNF in Apotosis Induction

In Section 1.1, a cooperative effect was observed between TNF andnaltrindole in inducing apoptosis. Because IκB abolished this apoptoticinduction, NF-κB was implicated in this apoptotic induction pathway.

In WO96/06863 it was shown that cytoplasmic and secretory proenkephalinrepress apoptosis in at least some cell types. Here we show thatsecretory proenkephalin can also act to promote apoptosis in concertwith TNF. Importantly, we also show that the cooperation betweensecreted proenkephalin and TNF is prevented by inhibition of NF-κBactivation, using IκB.

FIG. 2 provides a quantitative representation of FACS profiles from 293cells treated for 48 hours with a non-lethal concentration of TNF (5ng/ml) or transiently transfected with 5 μg of plasmid DNA encodingproenkephalin destined for the secretory pathway; in both cases noinduction of apoptosis is observed. However, when proenkephalin isoverexpressed in combination with TNF (bars as shown) there is a markedincrease in the percentage of apoptotic (sub-G1) cells diagonal-hatchedbars and a concomitant reduction in viable cells (stippled andhorizontal-hatched bars). The cooperation between proenkephalin and TNFis inhibited by co-transfection of 5 μg of plasmid DNA encoding theNF-κB inhibitor, IκB (bars on extreme right of graph) there is arecovery in the viable cells with a greater than 50% reduction in thepercentage of sub-G1 (apoptotic) cells. Notably, IκB alone does notinduce death; this indicates that opioids are not inducing apoptosis byproviding a “global” inhibition of NF-κB activation but instead, areswitching NF-κB into an apoptotic mode.

The ability of secretory proenkephalin to cooperate with TNF in an NF-κBdependent manner to induce death in 293 (transformed human kidney) cellsis therefore analogous to that seen with opioid-like compounds.

1.3 Opioid-Like Receptor Ligands Cooperate with TNF to Induce Apoptosisin Human Cancer Cells

In Sections 1.1 and 1.2, we have shown a cooperative effect between TNFand an opioid-like receptor ligand and between TNF and proenkephalin.

The cooperation between opioid-like receptor ligands and TNF is evidentin all human cancer cell lines we have tested so far including lung andcolon cancer. Here we show (FIG. 3) the effect of 48 hours treatmentwith the natural opium alkaloid noscapine at 10⁻⁴ M on p53 null lungcancer (H1299) cells; 39% of cells are apoptotic but there are stillmany viable cells. However, when noscapine is combined with TNF at 5ng/ml (which on its own has no effect on cell viability) there is amarked reduction in viable cells and a marked increase in apoptoticcells (to 74% of the total cell population). Importantly, H1299 cellslack functional p53 protein and therefore the cooperation with TNF doesnot require p53, which would be consistent with dependence on NF-κB.

FIG. 4 illustrates apoptotic death induced by noscapine and TNF in humancolon cancer (HT29) cells. In this particular cancer cell type,noscapine on its own at 10⁻⁴M induces apoptosis in 67% of the cellpopulation; there is an additional increase when TNF is added.

The cooperative effect of opioid-like ligands and TNF on the NF-κBdependent induction of apoptosis has been shown for different ligands ondifferent tumour cell lines. This cooperation between opioids and TNF,which is dependent on NF-κB, indicates that other anti-tumour therapieswhich are associated with NF-κB activation such as ionising radiation,nocodazole, etoposide, and daunorubicin will be enhanced by opioids.

1.4 Opioids and Proenkephalin (Nuclear and Secretory) Cooperate with theRelA(P65) Subunit of NF-κB to Induce Death

In the previous examples, opioid-like agents were shown to switch NF-κBthat had been activated by TNF into a pro-apoptotic activity. Theinteraction between proenkephalin and NF-κB was investigated further inthis example.

NF-κB is composed of subunits in different dimeric combinations. TheRelA(p65) subunit of NF-κB is dysregulated in inflammatory diseases suchas arthritis, and is downregulated on successful treatment (Mandel etal. 1995 Arthritis and Rheumatism Vol 38, pp 1762-1770; Marok et al.1996 Arthritis and Rheumatism Vol 39 pp 583-591; Tsao et al. 1997Clinical Immunology and Immunopathology Vol. 83 pp 173-178); RelA(p65)is also dysregulated in atherosclerosis, a disease characterised byfeatures of chronic inflammatory processes (Brand et al. 1996 Journal ofClinical Investigation Vol 97 pp 1715-1722; Bourcier et al. 1997 Journalof Biological Chemistry Vol. 272 pp 15817-15824); abnormal expression ofRelA(p65) also occurs in Alzheimer's disease (Terai et al. 1996 BrainResearch Vol 735 pp 159-168; Kitamura et al. 1997 Neuroscience Letts Vol237 pp 17-20) Dysregulation of RelA(p65) would be generally expected toprovide an anti-apoptotic drive (Beg et al., 1995 Nature Vol 376 pp167-170). NF-κB is also constitutively activated (RelA(p65) istranslocated to the nucleus) in at least some tumours such as Hodgkinslymphoma and late stage breast cancer. We therefore determined whetherproenkephalin and opioids would provide a pro-apoptotic switch tooverexpressed RelA(p65), as an experimental model of diseases whereRelA(p65) is dysregulated. If so, this would allow us to predict thatopioids would terminate or at least attenuate the inflammatory processeswhich are central to many major diseases.

In this example we show that overexpressed nuclear and secretoryproenkephalin cooperate with overexpressed RelA(p65) to induceapoptosis. FIG. 5 shows a series of phase micrographs of 293 cellstransiently transfected with DNA encoding nuclear proenkephalin (PE) onits own (top left), in combination with the RelA(p65) subunit of NF-κB(top right), and in triple combination with RelA(p65) and p53 (bottomright). Nuclear proenkephalin (PE) in combination with overexpressedRelA(p65) (NF-κB) produces dead, shrunken apoptotic cells (top rightpanel); in contrast, proenkephalin, or NF-κB alone (left hand panels)shows no significant death. This Figure also illustrates the more markedeffect when a triple combination of proenkephalin, RelA(p65) and p53 isused which exemplifies the potential of combination approaches usingopioids together with activators of both NF-κB and p53; it also suggeststhat opioids have the potential to override the reciprocity betweenNF-κB and p53 (Neil Perkins, unpublished). Secretory proenkephalin andopioid-like compounds also cooperate with RelA(p65) in apoptoticinduction (see example below for haloperidol).

In the context of anti-tumour therapy, the ability of opioids tocooperate with the RelA(p65) subunit of NF-κB indicates that opioidswill cooperate generally with activators of NF-κB which will includecompounds which were hitherto alien to the arena of tumour therapy; inparticular, we propose that microtubule depolymerising agents (such asnocodazole, colchicine, podophyllotoxin, vinblastine), which activateNF-κB as a consequence of this, would offer powerful combinatorialpossibilities. The ability of opioids to cooperate generally withactivators of NF-κB also demonstrates the potential for cooperation withinflammatory mediators and cytokines such as lipopolysaccharide,lymphotoxin-α, interferon-g; also anti-CD40 antibodies, CD40 ligand etc(see table above; from Siebenlist et al 1994 Annu Rev Cell Biol Vol. 10pp 405-455), using the agents at low doses to minimise pro-inflammatoryor other harmful side effects. Importantly, some of these agents (suchas CD40 ligand) activate NF-κB in a cell type specific manner and sowould target anti-tumour effects to the cell type in which themalignancy has arisen such as lymphoid cells.

2. Death Induced by Opioid Receptor Ligands is Itself Partly Dependenton NF-κB Activation

Examples 1.1-1.4 indicated that opioids had the ability to provide a“pro-apoptotic switch” to NF-κB activated by agents such as TNF. Thisfinding led us to address whether opioids may themselves activate NF-κBor induce a pro-apoptotic switch to constitutively activated NF-κB intumour cells, leading to death. The following examples demonstrate thatall the opioid-like compounds tested which induce apoptosis (includingnaltrindole, trans-U50488, noscapine, and sigma ligands such ashaloperidol) are indeed at least partly dependent on NF-κB activationfor their effect.

FIG. 6 depicts percentages of apoptotic compared with viable 293(transformed human kidney) cells following treatment for 48 hours withnaltrindole or trans-U50488 (middle left); in both cases there is amarked induction of sub-G1 (apoptotic) cells and a marked reduction inviable cells; in contrast, opioid treatment of cells transfected withmutant “super-repressor” IκB, to prevent NF-κB activation, fails toinduce death. It is important to note that this apparent completeprotection from death does not indicate exclusive dependence on NF-κBsince the p53 pathway is functionally inactivated in these cells by theadenoviral gene products E1B 19K and 55K. However, the lack of relianceon p53 for the induction of death by opioids makes p53 null cancer cellsa particularly attractive target for apoptosis induction according tothe various aspects of this invention, since these cells are moreresistant to anti-tumour therapies such as ionising radiation and DNAdamaging drugs. Apoptosis is therefore induced at least partly throughan NF-κB mediated pathway.

The ability of other activators of NF-κB such as TNF to enhance thedeath effect of opioids indicates that opioid activation of NF-κB is notsaturating. However, since there is a dearth of data on the state ofNF-κB activation in tumour cells, we cannot at this stage confidentlydistinguish whether opioids are themselves activators of NF-κB or areswitching pre-existing activated NF-κB into an apoptotic mode; however,the outcome would be the same.

These examples show that opioid-like compounds achieve their apoptoticeffect through an NF-κB dependent mechanism. This is shown, in thepresent application, for several different classes of opioid-likecompound (including naltrindole, trans-U50488, noscapine and sigmaligands such as haloperidol).

3. Tumour Cells in which NF-κB is Constitutively Activated at HighLevels, Such as Hodgkins Lymphoma Cells, are Particularly Susceptible tothe Effects of Opioids; Control Lymphoid Cell Lines are Unaffected.

Because opioid-like compounds achieve apoptotic induction through NF-κB,as shown in the previous examples, they may be particularly suitable forcertain therapeutic applications.

Few studies have addressed how widely NF-κB is dysregulated in tumourcells. However, there are some tumour types where NF-κB has been shownto be constitutively activated; for example, in Hodgkins lymphoma cellsNF-κB is deregulated at high levels and fails to respond to activatorsof NF-κB such as CD40 ligand (Wood et al. 1998 Oncogene Vol. 16 pp2131-2139). We therefore addressed whether Hodgkins cells would be moresusceptible to the effects of opioid-like agents, in the absence ofconcomitant activators of NF-κB.

FIG. 7 a illustrates a FACS profile of L428 Hodgkins lymphoma cellstreated for 48 hours with either noscapine or naltrindole. Both agentsat 10⁻⁴M induce the majority of the cell population to apoptose; even at10⁻⁵M a substantial amount of apoptosis is seen and there is in additionevidence of mitotic arrest (increase in percentage of G2/M cells). Thus,Hodgkins lymphoma cells are particularly susceptible to the effects ofopioids; the degree of death induction in Hodgkins cells by opioid-likereceptor ligands administered alone, is approximately the same as thatinduced by opioids in combination with TNF in other cancer cell lines(see for example lung cancer cells treated with noscapine at 10⁻⁴M inthe present and absence of TNF, FIG. 3). These data therefore indicatethat opioid-like compounds can confer a pro-apoptotic switch topre-existing activated NF-κB even when it is deregulated at high levels.They also demonstrate comparable activity of naltrindole and noscapine.

FIG. 7 b illustrates of control B lymphoid cells, which lack deregulatedRelA(p65), treated for 48 hours with noscapine or naltrindole.Interestingly, untreated control B cells have a high level(approximately 50% of cells) of basal apoptosis (sub-G1) which reflects“appropriate” apoptosis; this “appropriate” apoptosis is absent inuntreated Hodgkins lymphoma cells (FIG. 7 a) which indicates lessreliance on external factors. Many haemopoietic cell lines are extremelysensitive to their supply of external survival signals and readilyundergo apoptosis in tissue culture as they are in an alien environment.It is noteworthy that neither noscapine nor naltrindole affect this“appropriate” apoptosis (approximately 50% cells remain apoptotic);also, the proportion of viable, cycling cells is very similar to that inuntreated cell populations. These data therefore provide powerfulevidence that opioid-like compounds spare proliferating non-tumour cellpopulations in both cell division cycle arrest and apoptotic effects.

These results demonstrate the preferential effect of opioid-likecompounds on tumour cells compared to non-tumour cells.

4. Evidence for Induction of Mitotic Arrest Prior to Apoptosis byOpioid-Like Compounds.

The idea that apoptosis is an aborted mitosis is not generally accepted,as discussed above; it is certainly not a common mechanism of apoptosisinduction. However, we have noted that when tumour cells are induced toapoptose in response to all the opioid-like compounds we have tested,this is preceded by an induction of cell division cycle arrest; this isalso a feature of cells treated with sub-apoptotic doses of opioids whencombined with TNF (see for example haloperidol plus TNF, FIG. 9 a).Induction of mitotic arrest and apoptosis by noscapine has also beendescribed by Ye et al (1998 PNAS, see above).

FIG. 8 depicts a FACS scan of H1299 cells treated with naltrindole whichshows a marked induction of apoptosis after 36 hours (bottom panels);however, 12 hours earlier (at 24 hours), this was preceded by theappearance of a G2/M peak (middle panels) which indicates thepossibility of mitotic arrest preceding entry into apoptosis. However,the simultaneous demonstration of at least a proportion of cells inapoptosis at this same time point suggests that this may not be anexclusive mechanism of apoptosis induction by opioids. Nonetheless, itis an otherwise uncommon profile which as far as we can tell is sharedby all pro-apoptotic opioid-like compounds. The advantage of thiscombination of mitotic arrest and apoptosis indicates a back-upmechanism if apoptosis fails; furthermore, as shown in the precedingsection, proliferating non-Hodgkins cells do not arrest in mitosis whichindicates further cell cycle-mediated potential for preferential effectson tumour compared to non-tumour cells.

This example shows the link between opioid induced apoptosis and mitoticarrest.

It is important however to emphasise that induction of mitotic arrest byopioids themselves is not a pre-requisite for apoptosis induction;indeed, WO96/06863 described a powerful combinatorial effect betweennocodazole, which arrests cell in mitosis, and opioids. As describedabove, nocodazole depolymerises microtubules and in so doing, activatesNF-κB; the role of opioids in this case is likely to be the provision ofa pro-apoptotic switch to NF-κB activated through themicrotubule-destabilising effect of nocodazole. It is important howeverto emphasise that the combination of microtubule depolymerising agentswith opioids could not have been predicted as a general combinatorialmechanism at the time WO96/06863 was filed; this is because NF-κBactivated as a consequence of microtubule depolymerisation would begenerally surmised to limit any apoptotic effect.

A summary therefore of possible ways in which opioids cooperate withNF-κB in the induction of death, which are not mutually exclusive andmay even act in cooperation, would be as follows:

1. Opioid-like receptor ligands become translocated to the cell interiorthrough binding to cell surface molecules, and are thence delivered tomicrotubules where a depolymerising effect induces mitotic arrest andalso activates NF-κB. Opioid-like agonists or antagonists then modulatethe outcome of NF-κB activation by providing a pro-apoptotic switch inthe way described.

2. Other agents which depolymerise microtubules such as nocodazole (andwhich may possibly synergise with opioid effects mediated throughdifferent binding sites on tubulin) activate NF-κB which is thenconverted into apoptotic mode by opioids.

3. Opioid-like agents provide a pro-apoptotic switch to NF-κB activatedby agents which act independently of microtubules, of which there aremany: TNF is one example and the cooperation of opioids with RelA(p65)exemplified the potential of multiple NF-κB activators.

4. Opioid-like agents themselves may activate NF-κB independently ofmicrotubules, through signal transduction events mediated at the cellsurface or by binding to intracellular opioid receptors such as havebeen reported in the cell nucleus (Ventura et al. 1998 J. Biol. Chem.Vol 273 pp 13383-13386); these events would lead tophosphorylation-mediated degradation of the inhibitor protein IκB andtranslocation of p65RelA (NF-KB) to the cell nucleus (Verma et al 1995Genes Dev. Vol. 9 pp 2723-2734). Opioid-like agents would in turnmodulate the outcome of NF-κB activation to end in an apoptotic fate.

5. Sigma Receptor Ligands, Halolperidol and Rimcazole, are PowerfulInducers of Apoptosis in Tumour Cells and Employ the Same Mechanism asOther Opioid-Like Agents.

We then determined whether sigma receptor ligands would induce apoptosisin tumour cells, by the same mechanism as the other opioid-like receptorligands. In these examples, we show that ligands which have antagonisticeffects at the sigma receptors, haloperidol and rimcazole, are potentinducers of NF-κB dependent apoptosis in all tumour cells we have testedso far. These data suggest that a sigma-like receptor mediates asurvival advantage on which tumour cells have come to preferentiallydepend.

As shown herein, in Example 7, other sigma receptor types mediate deathrather than survival. Agonistic ligands for these sigma receptor typeswould also be pro-apoptotic.

FIG. 9 a illustrates cooperativity between haloperidol and TNF in theinduction of apoptosis in p53 null human lung cancer cells; thiscooperativity is particularly marked at the 10⁻⁵M dose level ofhaloperidol (4% apoptosis is converted to 59% apoptosis when the twoagents are combined)

FIG. 9 b demonstrates that haloperidol also cooperates powerfully withthe RelA(p65) subunit of NF-κB. This experiment was carried out in the293 cell line to achieve high levels of p65 transfection efficiency.There is a powerful cooperation between haloperidol at 10⁻⁵M combinedwith RelA(p65) (47% of cells are apoptotic compared with 2-3% apoptosiswith either treatment on its own). Thus, a truly synergistic apoptoticeffect is revealed by the combination of haloperidol and RelA(p65) (asit is also with haloperidol and TNF).

FIG. 9 c demonstrates the dependence of haloperidol-induced death onNF-κB (in 293 cells, where p53 is functionally inactivated); sub-G1apoptotic cells are markedly reduced (from 58% to 5% of the total cellpopulation) when plasmid DNA encoding non-degradable mutant IκB istransfected prior to haloperidol treatment.

Rimcazole, another more specific sigma receptor antagonist which lacksthe dopamine antagonist effects of haloperidol, produces the sameeffects. Interestingly, both haloperidol and rimcazole cooperate withtrans U50488.

FIGS. 10 a and 10 b illustrate the induction of apoptotic death in lungcancer (H1299) cells by rimcazole. FIG. 10 a shows cooperativity betweena sublethal dose of TNF (tumour necrosis factor) and a sublethal dose ofrimcazole (lower left hand panel) to produce significant apoptosis(increasing from 4% to 61% of cells) and a marked reduction in viablecells.

FIG. 10 b depicts cooperativity between a sublethal dose of thetrans-isomer of U50488 (0.1 mM; sublethal at high cell density—seeWO96/06863); and a sublethal dose of rimcazole (0.01 mM); these agentsin combination produce significant apoptosis (4-6% apoptosis increasingto 67%). Hence, a kappa opiate receptor ligand synergises with a sigmareceptor ligand in apoptosis induction.

These examples show that sigma ligands mediate apoptotic inductionthrough the same NF-κB dependent pathway as other opioid-like agents.Cooperativity with the NF-κB activating agent TNF, inhibition by theNF-κB inhibitor IκB and synergy with the NF-κB subunit RelA(p65} arefeatures of apoptotic induction through this pathway.

6. Hodgkins Lymphoma Cells, in which NF-κB is Constitutively Activatedat High Levels, have an Increased Sensitivity to the Apoptotic Effectsof Sigma Ligands; Control B Cells are Unaffected

FIGS. 11 a and 11 b illustrate a FACS profile of Hodgkins lymphoma cellstreated for 22 hours with doses of haloperidol or rimacazole from 10⁻⁴Mto 10⁻⁶M. In all cases, there is marked induction of apoptosis (34% to47% of cells are apoptotic even with micromolar concentrations ofcompounds). Thus sigma ligands are particularly potent at inducingapoptosis in cells in which RelA(p65) is deregulated.

In contrast, FIG. 11 c depicts control B cells which lack deregulatedRelA(p65) and are unaffected by either rimcazole (FIG. 11 c) orhaloperidol (not shown). Once again, untreated cells show a significantlevel of ‘appropriate’ apoptosis due probably to deprivation of externalsurvival signals; neither rimcazole nor haloperidol affect this‘appropriate’ apoptosis. Furthermore, the sigma ligands do not affectthe cell cycle profile of the viable control cells. Thus, this is a veryclear demonstration of a marked preference of sigma ligands to induceapoptosis in authentic human tumour cells but not in a controlpopulation of proliferating cells.

Together, these data provide powerful evidence to support a commonmechanism of apoptosis induction which is shared by ligands of classicaland non-classical opioid receptors, including the sigma receptor class.

7. Enhanced Sensitivity of Hormone Unresponsive (Advanced Stare) BreastCancer Cells to Sigma Ligands, Compared to Hormone Responsive BreastCancer Cells.

As described above, constitutive activation of NF-κB has been linked toprogression of breast cancer to hormone-independent growth (Nakshatri etal. Mol. Cell. Biol. 1997 Vol 17 pp 3629-3639); the cell lines describedin this article were MCF 7 (as a model of hormone responsive breastcancer) and MDA (MB) 231 (as a model of hormone unresponsive, advanced,breast cancer). FIG. 12 illustrates substantially greater induction ofapoptosis in MDA 231 cells compared to MCF 7 cells with the sigmaligand, rimcazole. In untreated cells of both types, there is negligibleapoptosis. However, when treated with rimcazole at 0.001 mM, 25% of thetotal MDA 231 cell population is apoptotic after 48 hours treatment;this compares with only 6% apoptosis in MCF 7 cells after the sametreatment regime and in the same culture conditions. There is also anincrease in the proportion of G2/M cells (62% compared to 32% in MCF 7cells) in rimcazole-treated MDA 231 cells which indicates induction of aG2/M cell division cycle arrest.

Thus, rimcazole induces substantially more apoptosis (25% compared to6%) and a greater degree of cell division cycle arrest (62% compared to32%) in hormone unresponsive breast cancer cells compared to hormoneresponsive breast cancer cells. Sigma ligands may therefore beparticularly applicable to advanced stage cancers. In this particularmodel, a component of enhanced susceptibility may be due to the presenceof constitutively activated NF-κB at high levels, which is “switched”into pro-apoptotic mode by the sigma ligand. It also remains possible,as we proposed earlier (page 2 of this document and WO96/06863), thattumour cells progressively acquire a preferential reliance onsigma-mediated survival due to a progressive loss of “back-up” survivalmechanisms. This indicates a wider applicability of sigma ligands toadvanced cancer even where NF-κB is not implicated.

8. Sigma Ligands Cooperate to Induce Apoptosis in Advanced Breast CancerCells.

We propose herein that sigma receptors may mediate death as well assurvival; this an unexpected extension of the disclosure of WO96/06863that opioid-like pathways in general mediate both death and survival andthat tumour cells are therefore unduly poised on an opioid-mediatedknife edge between life and death.

FIG. 13 illustrates a representative experiment in which cooperativeinduction of apoptosis occurs when two sigma ligands are combined:rimcazole and cis-U50488 (which is distinct from the kappa agonisttrans-U50488). In this series of experiments another model of advancedbreast cancer was used, MDA (MB) 468, to determine whether sigma ligandswould be generally effective against hormone unresponsive breast cancer.

Rimcazole at a concentration of 0.01 millimoles per liter inducesapoptosis in 79% of the MDA MB 468 cell population approximately 48hours after addition of the compound. At a lower concentration ofrimcazole (0.001 mM) 16% of cells are apoptotic at the same time point;however, if this is combined with cis-U50488 at 0.1 mM (which on its owninduces apoptosis in 11% of the cell population), 83% of the cells arenow induced to apoptose; this indicates synergistic death induction whendifferent sigma ligands are combined.

Rimcazole is generally viewed as an antagonistic sigma ligand whereascis-U50488 is generally viewed as an agonistic sigma ligand. However, itshould be stated that, until the signal transduction events whichmediate the regulation of apoptosis through the sigma receptor have beendefined, the precise categorisation of ligands as either antagonistic oragonistic must remain provisional. That said, these data indicate thatmore than one sigma receptor (or more than one binding site on the samereceptor macromolecule) is involved in regulating apoptosis, and thatthere is a close functional relationship between these sites.

9. Sigma Ligands Cooperate with p53 in Apoptosis Induction

We propose herein also that sigma ligands could be combined withactivators of p53. Many activators of the endogenous p53 pathway (suchas etoposide, and ultraviolet and gamma irradiation) induce concomitantactivation of the NF-κB pathway. Activation of the p53 pathway causesreduced degradation of the p53 protein which leads to an increase in p53protein levels within the cell. Therefore, to simulate activation of thep53 pathway in isolation, we used three different model systems in whichthe p53 protein is conditionally or transiently overexpressed.

FIG. 14 a illustrates breast cancer (MCF 7) cells into which atemperature sensitive mutant form of p53 has been introduced (MCF7::p53^(val135)). When these cells are grown at the permissivetemperature (32 degC.) p53 adopts a wild-type conformation; but whengrown at the restrictive temperature (37 degC.) the p53 protein adopts amutant, inactive conformation.

FIG. 14 a (upper 4 panels) illustrate cells grown at 37 degC. (at whichtemperature the p53 protein is largely inactive) in the presence andabsence of rimcazole. Apoptosis is induced with rimcazole at high dose(0.1 mM) but the cells are largely unaffected at doses of 0.01 mM andbelow.

To restore the conformation of the p53 protein to, or closer to, itswild-type state the temperature at which the cells are cultured isreduced to 32 degC.; in the absence of rimcazole this has little effectbut there is a marked induction of apoptosis (phase micrographs showtypically rounded, shrunken cells) with doses of rimcazole which have noeffect at the higher temperature (see for example 0.001 mM).

To confirm cooperativity between sigma ligands and the p53 protein,another system in which p53 is under inducible control was used (FIG. 14b). In this model system, (Saos-2::p53^(teti)), p53 cDNA driven by atetracycline-inducible promoter has been introduced into a humanosteosarcoma cell line which is ordinarily p53 “null”. With increasingdoses of the tetracycline analogue, doxycycline, p53 can be induced toprogressively higher levels. At high doses of doxycycline, andconcomitant high levels of the p53 protein, apoptosis is induced.However, at low doses of doxycycline the small increase in p53 proteinlevels induces a small percentage of cells (4% of the total populationin this representative experiment—FIG. 14 b) to undergo apoptosis.Similarly, a low dose of rimcazole (100 nM) induces approximately 5% ofthe cell population to apoptose. However, when rimcazole at 100 nM iscombined with doxycycline treatment at a concentration of 0.02 μg/ml,there is now a synergistic induction of apoptosis such that 85% of cellsare apoptotic. In wild-type Saos-2 cells which lack p53 protein, thecombination of low dose rimcazole and doxycycline has no effect; thisimplicates induced p53 as the mediator of cooperativity betweenrimcazole and doxycycline in Saos-2::p53^(teti) cells.

In FIG. 14 c a third model system is illustrated: this illustratescooperativity between rimcazole and p53 when transiently overexpressedin 293 (transformed human kidney epithelial cells). p53 (at this lowdose) and rimcazole alone (at 10 mM concentration) induce only 1% of thecell population to apoptose; however, in combination, there is markedsynergy such that 75% of the cell population are now induced toapoptose. Importantly, this also illustrates a biological effect ofrimcazole when administered at a low concentration (10 nM) whichindicates that authentic sigma receptors are involved in mediating theapoptotic effect.

Together, these data indicate that sigma ligands (or other opioid oropioid-like mediators of apoptosis) could be combined with activators ofp53 such as gamma irradiation or genotoxic chemicals in the treatment oftumours which possess wild-type p53 protein; and in combination withgene therapy to reintroduce p53 into tumours which lack functional p53protein; and in combination with agents which restore p53 conformationto tumours which possess mutant p53 protein.

10. Sigma Ligands Inhibit the Growth of Human Breast CarcinomaXenografts in Athymic Mice. Materials and Methods

MDA MB 468 human breast carcinoma cells (described above, Section 7, asa model of hormone-insensitive disease) were grown in tissue culture to70% confluence, harvested with trypsin:EDTA, washed, resuspended in DMEMand injected subcutaneously bilaterally in the flanks of 60 six-week oldfemale Onu/Onu mice at 2×10⁶ cells/site.

Experimental groups N mice 1. Control (water) 12 2. Rimcazole (10mg/kg/day) 8 3. Haloperidol (10 mg/kg/day) 8 4. cis-U50488 (10mg/kg/day) 8

All drugs were dissolved in sterile water for injection and frozen as ˜2ml aliquots, except haloperidol which was stored as a powder at RT andmade up fresh each day in 45% cyclodextrin. The mice were housed infilter boxes in Maximiser laminar flow cabinets and fed sterilized foodand water. All procedures were carried out in class 1 laminar flow hoodsusing sterile equipment and reagents. The mice were dosed daily by anintraperitoneal route (combined therapies dosed simultaneously) and bodyweights and tumour measurements recorded at frequent intervals. Tumourswere measured with vernier calipers across two perpendicular diametersand results expressed as tumour volume calculated according to theformula V=4/3π[(d1+d2)/4]³.

The experiment was terminated on day 45 when tumours were excised,dissected free of surrounding normal tissue and weighed. Differences ingrowth were compared using the Mann-Whitney U test. Tumour samples werefixed in Methacarn for later histological examination.

Results

Mice tolerated the therapy well, with no ill effects except fortransient sleepiness in the haloperidol treated groups. This isconsistent with in vitro data described in Section 5 (FIG. 7) thattumour cells are preferentially affected by sigma ligands compared withnormal cells. The tumour “take rate” (i.e. incidence of tumoursdeveloping from the total number of injected sites) was, as expected,less than 100%. Since therapy was initiated on day 0, preventingexclusion of “non-takes” prior to randomisation, the decision was takento include analyses of both a) all tumour loci (FIG. 15 b) and b)excluding sites which were negative at the second measurement on day 17(FIG. 15 c). The results are shown in FIG. 15 a (mean tumour volume) andFIG. 15 b&c (mean tumour weight +/−standard deviations). Mann Whitney UTests (2 tailed) were carried out to compare tumour weights in treated(rimcazole, cis-U50488 or haloperidol) groups with tumour weights in thecontrol group. Statistically significant effects (P value less than0.05) are indicated by bars with asterisks (***).

Analysis of the results (based on tumour weights at excision) determinedthat rimcazole resulted in significant tumour growth inhibition(regardless of whether analysis included all or selected tumours). Ifthe “non-takes” (did not appear to grow tumours) were excluded, cisU50488 also showed significant tumour growth inhibitory effects.Haloperidol also showed growth inhibition. These data confirm that sigmaligands, antagonistic and agonistic are potent anti-tumour agents invivo, with no symptomatic evidence of toxicity. MDA MB 468 cells possessmutant p53 protein and are characteristic of advanced stage human breastcancer. Application of sigma ligands is therefore particularlyapplicable to this type of cancer.

APPENDIX 1

IUPAC chemical names for 1) named agents which induce apoptosis and 2)prototypic sigma ligands for assays to define sigma agents allobtainable from Sigma Ltd (Poole, Dorset, UK).

Naltrindole4,8-methanobenzofuro[2,3-a]pyrido[4,3-b]carbazole-1,8a(9H)-diol,7-cyclopropylmethyl)-5,6,7,8,14,14b-hexahydro

(CAS registry numbers 111469-81-9)

Trans-U50488trans-3,4-Dichloro-N-Methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)benzene-acetamide

(CAS registry numbers 83913-06-8)

Noscapine(S-(R*,S*))-6,7-Dimethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo(4,5-g)isoquinolin-5-yl)-1(3H)-Isobenzofuranone

(CAS Registry Numbers 128-62-1)

Haloperidol4(4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl)-1-(4-fluorophenyl)-1-Butanone

(CAS Registry Numbers 52-86-8)

Rimcazole (BW234U)cis-9-[3-(3,5-Dimethyl-1-piperazinyl)propyl]-9H-carbazole

(CAS registry numbers 75859-04-0).

(+)-Pentazocine(+)-(2alpha,6alpha,11R*)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(3-methyl-2-butenyl)-2,6,-Methano-3-benzazocin-8-ol

(CAS registry numbers 359-83-1)

DTG 1,3-Di(2-tolyl)guanidine

(CAS registry numbers 97-39-2)

cis-U50488 is supplied from Sigma Ltd (Poole, Dorset, UK).

Tumour necrosis factor (TNF) is supplied by TCS Biologicals LTD(Buckingham, Bucks, UK).

Nocodazole, etoposide, colchicine, daunorubicin, podophyllotoxin andvinblastine are all supplied by Sigma Ltd (Poole, Dorset, UK).

Expression vectors encoding proenkephalin, p65 (RelA), IκB and p53 areall obtainable from MSI/WTB Complex, University of Dundee, Dundee, UK.

Agonistic anti CD40 antibody provides a means of stimulating B cellsthrough CD40. A suitable antibody is available from PharMingen a BectonDickinson Company, Becton Dickinson GmBH, HQ PharMingen Europe Cat. No.33070D.

1-47. (canceled) 48: A method for treatment of cancer in a patient inneed of such treatment, said method comprising contacting the cancercells with a therapeutically effective amount of rimcazole. 49: Themethod of claim 48, wherein said cancer cells are contacted with aconcentration of rimcazole of 10 micromolar or less. 50: The method ofclaim 48, wherein said cancer cells are contacted with a concentrationof rimcazole of less than 1 micromolar. 51: The method of claim 48,wherein said cancer is selected from the group of breast cancer, lungcancer, or Hodgkin's lymphoma. 52: The method of claim 48, furthercomprising the administration of an agonist for a kappa receptor. 53:The method of claim 48, further comprising the administration of atleast one additional sigma receptor ligand. 54: The method of claim 48,wherein the cancer cells are p53 null tumor cells. 55: The method ofclaim 48, wherein the cancer cells are characterized by the presence ofconstitutively activated NF-kappaB in cells where it would not normallybe found. 56: A method for the preferential induction of apoptosis in apopulation of tumor cells in a human compared to a population ofnon-tumor cells in a human, said method comprising exposing the firstand second population of cells to rimcazole. 57: The method of claim 56,wherein the cells are exposed to rimcazole at a concentration of 10micromolar or less. 58: The method of claim 56, wherein the cells areexposed to rimcazole at a concentration of less than 1 micromolar. 59:The method of claim 56, further comprising exposing the cells to anagonist for a kappa receptor. 60: The method of claim 56, furthercomprising exposing the cells to at least one additional sigma receptorligands. 61: The method of claim 56, wherein the cells of the populationof tumor cells are p53 null tumor cells. 62: The method of claim 56,wherein the cells of the population of tumor cells are characterized bythe presence of constitutively activated NF-kappaB in cells where itwould not normally be found.